Rickettsia felis was originally identified in the United States as a human pathogen in 1991 and is now associated with human infection in North and South America, Europe, Africa, Asia, and Oceania. Our ultimate goal for this research is to elucidate the biological and molecular mechanisms that are critical to rickettsial transmission by fleas in order to better understand the epidemiology of flea-borne rickettsial diseases and identify novel points of intervention. The results of the proposed studies will determine if R. fels utilizes multiple mechanisms for rapid horizontal transmission between fleas independent of a rickettsemic vertebrate host. This is a new paradigm for vector-borne rickettsial diseases. Also, the probability that a novel rickettsial response occurs in the arthropod host, and is somehow associated with rickettsial transmission, has long been a tenant of vector-borne rickettsial diseases; however, this has never been tested in an actual transmission system. The experimental focus of this application is to delineate horizontal transmission mechanisms through comparative analyses three distinct rickettsial strains in two different arthropod vectors, cat fleas and rat fleas. The rickettsial-derived molecules underlying the transmission of R. felis in flea hosts are not known. Studies will also employ rickettsial mutagenesis to identify transmission determinants in a flea transmission system. Two limiting factors for vector/disease management and the barriers to advancing the field are the scant knowledge of 1) basic transmission biology of R. felis and, 2) the rickettsial-derived determinants of transmission. Through completion of the specific aims outlined in this application, these studies will overcome the hurdles by assessing rickettsial transmission by fleas (Specific Aim 1) and through identification of Rickettsia-derived molecular constituents essential to transmission events (Specific Aim 2). Thus, this is a multifaceted approach to decipher the vector and pathogen-associated factors essential to transmission and will provide a platform to examine other flea-borne bacterial pathogens. Aim 1. Delineate the mechanism by which Rickettsia felis is acquired and transmitted by arthropods. Stable vertical transmission of certain vector-borne pathogens eliminates the need for a vertebrate host; however, unless this transmission event is 100% efficient, additional horizontal amplification is required for maintenance of the pathogen in the environment. We will test the hypothesis that if vertical transmission of R. felis by fleas is not 100%, then horizontal transmission of R. felis must occur between fleas. The objective of this aim is to identify novel routes of R. felis acquisition by fleas and assess the intra- and inter-specific transmission of R. felis. Aim 2. Define the Rickettsia-derived molecular constituents of transmission by flea vectors. Vector-borne bacterial pathogens undergo essential molecular transformation in the vector prior to transmission to the vertebrate host. We will test the hypothesis that if R. felis expresses a distinct rickettsial protein profile that orchestrates infetion of arthropods and subsequent transmission, then disruption of key molecules will impede infection and/or transmission. To test this hypothesis, we will focus on (a) targeted mutagenesis in R. felis and (b) analysis of isogenic rickettsial mutants in flea-infection and transmission.